1. Field of the Invention
The invention relates to the synthesis of macrocyclic polyaminocarboxylate chelating agents which can be used for the stable attachment of radiometals to biological molecules such as proteins and antibodies. The conjugated chelates can be used for diagnostic and therapeutic applications.
2. Description of the Related Art
Macrocyclic polyaminocarboxylates such as 1,4,7,10-tetraazacyclododecane N,N',N",N'"-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane N,N',N",N'"-tetraacetic acid (TETA) are known to bind metals very tightly. ##STR1## DOTA and TETA were first prepared by J. F. Desreux (Inorg. Chem. 19:1319, 1980), by alkylation of 1,4,7,10-tetraazacyclododecane 1 and 1,4,8,11-tetraazacyclotetradecane 2 respectively with chloroacetic acid in base followed by acidification Which sometimes precipitates the crude product. The crude product was further purified on cation exchange resin.
U.S. Pat. Nos. 5,132,409, 5,049,667, 4,923,985, 4,678,667 and 4,639,365 describe the synthesis of derivatives of 1,4,7,10-tetraazacyclododecane 1 and 1,4,8,11-tetraazacyclotetradecane 2 which are converted to the corresponding derivatives of DOTA and TETA using haloacetic acid with methods similar to that of Desreux, Id.
The ability of DOTA and TETA to tightly bind metals is advantageously used in contrast agents for magnetic resonance imaging, e.g. gadolinium-DOTA described by Magerstadt et al., Mag. Res. in Med. 3: 808-812 (1986), and for the formation of stable radiometal labeled antibodies for diagnosis and/or therapy as described by Meares et al., Br. J. Cancer 62: 21-26 (1990). For chemical attachment to antibody, DOTA and TETA must be suitably functionalized. In order to attach DOTA and TETA to antibodies these ligands must be derivatized with a group which can react with a group on the antibody such as an amine or sulfhydryl group. Several lengthy synthetic routes have been proposed to functionalize the carbon backbone of DOTA and TETA. ##STR2##
A para-bromoacetamidobenzyl derivative of TETA 3 was prepared by M. K. Moi et al., Anal. Biochem. 148: 249-253 (1985) and U.S. Pat. No. 4,678,667 utilizing a low yield (about 12%) cyclization reaction. The para-bromoacetamidobenzyl derivative of DOTA 4 was also prepared by M. K. Moi et al., J. Am. Chem. Soc. 110: 6266-6267 (1988) and U.S. Pat. No. 4,678,667 using a higher yield intramolecular cyclization reaction. Compounds 3 and 4 were conjugated to antibodies via 2-iminothiolane as described by M. J. McCall et al., Bioconjugate Chem. 1: 222-226 (1990). Para-isothiocyanato-benzyl-DOTA and -TETA derivatives, compounds 5 and 6 were prepared by a different route whose key cyclization step utilized two acylation reactions under high dilution conditions as described by O. A. Gansow, Nucl. Med. Biol. 18: 369-381 (1991) and U.S. Pat. No. 4,923,985. Compounds 5 and 6 did not require treatment of the antibody with 2-iminothiolane for conjugation.
Another functionalized DOTA, 1-n-butylamino-DOTA, was prepared by Cox et al., J. Chem. Soc. Chem. Commun. 1989: 797-798 (1989). The key step in this route was a Richman Atkins cyclization. This compound was then further functionalized by reacting with the p-nitrophenyl ester of 2-vinyl-6-(4'-carboxy-3'-oxa-butyl)pyridine to give 7 which could then be attached to antibodies using 2-iminothiolane. ##STR3##
All the methods listed above for the preparation of functionalized DOTA or TETA require long multi-step syntheses containing difficult cyclization steps. Others have investigated shorter routes which start with the commercial macrocycles 1,4,7,10-tetraazadodecane 1 or 1,4,8,11-tetraazacyclotetradecane 2. For example, a route to 8 which leaves one nitrogen available for further functionalization has been described by Dischino et al., Inorg. Chem. 30: 1265-1269 (1991). The Dischino et al. method is similar to that of Desreux, Inorg. Chem. 19: 1319 (1980) discussed above, involves the alkylation of commercially available 1,4,7,10-tetraazacyclododecane 1 with chloroacetic acid and requires anion exchange purification. The synthesis of 9 which is DOTA functionalized on a carbon alpha to one of the carboxylates is described by Kline et al., Bioconj. Chem. 2: 26-31 (11991). The key step in the Kline et al. synthesis is the alkylation of 1 to give 10. The non-alkylated amine is then further functionalized to give 9.
A simpler method of functionalizing DOTA which sacrifices one of the carboxylates is to form the mixed anhydride with isobutylchloroformate to give 11. Poly-lysine-DOTA (Sieving et al., BioconJ. Chem. 1: 65, 1990) and biotin-DOTA (Wu et al., Nucl. Med. Biol. 19: 239, 1992) have been prepared by this method. A drawback of this method is that mixed anhydrides are very prone to hydrolysis and therefore these preparations cannot be stored for any length of time. ##STR4##
Previously described methods for the synthesis of DOTA and TETA have the disadvantages of requiring the use of haloacetic acid and require purification of the product by ion exchange. Other methods involve lengthy multi-step syntheses and difficult cyclization reactions and some yield unstable mixed anhydride product. Therefore, it would be highly desirable to provide a simple, high yield synthetic method for DOTA and TETA which can be coupled with a simple but effective functionalization reaction.
Furthermore, conjugation of DOTA and TETA chelates to antibody may be adversely affected by crosslinking. Crosslinking occurs when two or more active esters or mixed anhydrides are formed on each ligand and each active ester group/mixed anhydride reacts with a different antibody, thereby crosslinking the two antibodies. Crosslinking of antibodies produces dimers and oligomers which increases the molecular weight of the conjugate and results in an increase in non-target liver uptake of the labeled preparation. This can occur with the active ester or mixed anhydride methods since both are formed from the tetraacids. The key in these is to limit the formation of ester or mixed anhydride to one group per molecule. This is usually done by using excess DOTA or TETA when making the active ester or mixed anhydride. But since mixed anhydrides are readily hydrolyzed (split by the addition of water), and since a biological compound being conjugated is usually in water, water competes with the group being conjugated. Therefore, anhydrides are not very efficient. This disadvantage is shared by some of the prior art conjugation methods described above and also by the DTPA dianhydride method. Therefore it would be desirable to synthesize DOTA and TETA derivatized in a manner which allows chemical attachment to antibody with no cross-linking and gives immunoconJugates that have good serum stability and that produce good tissue distribution in vivo.